The genetic structure of populations is not necessarily reflected in the geographical proximity of individuals, because environmental gradients such as those of vegetation or climate can function as cryptic barriers to gene flow. We examined polymorphisms at nine microsatellite loci to determine and discuss whether a distinctive genetic structure was detectable in a spatially continuous population of the sika deer (Cervus nippon) on the Boso Peninsula of central Japan. Spatially explicit Bayesian analysis revealed that two genetically distinctive clusters exist in the Boso population. The spatial boundary of the two clusters approximately conformed to the border defined previously from a mitochondrial DNA dataset. By combining information on the geomorphic features surrounding the boundary and that on the lineage of 1970s population, we propose a schematic scenario for characterizing the population genetic structure to the present. The current population consists of genetically different lineages, and spatially discontinuous clusters have come into contact in the vicinity of a local road running along a steep-walled ravine that could act as principal barrier to gene flow. Biological factors such as distribution of vegetation and philopatric behavior might also have helped strengthen the cryptic genetic structure of the Boso population.